Hotspot in ferruginous rock may have serious implications in Brazilian conservation policy

A hotspot of subterranean Collembola in ferruginous rock caves and Mesovoid Shallow Substratum is revealed by the analysis of pseudocryptic diversity. The diversity is accessed by detailed description of chaetotaxy and slight variation in morphology of 11 new species of Trogolaphysa Mills, 1938 (Collembola, Paronellidae, Paronellinae) and the 50 previously recorded species of springtails from caves, using optical and electronic microscopy. When combined with recent subterranean surveys, our results show an important reservoir of cave diversity in the Mesovoid Shallow Substratum. Contrastingly the conservation policy for subterranean fauna in metallogenic areas in Brazil prioritizes the caves instead the cave species, which may be extremely detrimental to the fauna in the shallow subterranean habitats not accessible to humans.


Discussion
Ferruginous mesovoid shallow substratum. The iron ore deposits in Brazil present a semi continuous covering layer of fragmented hematite and lesser components cemented by limonite, called Canga. It is formed by weathering and lixiviation, and produce a labyrinthic complex of subterranean spaces, crevices, and tiny underground connections, depicting a habitat that is analogous to the MSS 12 .
In temperate zones the MSS plays a role as refuge for arthropod fauna, mainly at high altitudes where the cold weather can eliminate all the ectothermic fauna from the surface 10,11 . Similarly, seasonal migration movements are observed in the MSS for different taxa as response to hot dry summer 20,21 . When troglobitic fauna is concerned the MSS has a different role, cave restricted Collembola showed higher underground dispersal capacity than troglophiles 22 , therefore, the MSS can connect neighboring caves systems and extend their distribution range.
In Brazilian metallogenic rock, cave species richness is higher than in any other lithology 12 . The cave Collembola found in Brazil corroborates this assumption, from the total of 61 known cave species, one troglobite was recorded from sandstone caves and one from granitic cave, 18 species were recorded from limestone caves (all troglobites), and 44 species from iron caves and MSS (31 troglobites). Three troglobitic species were recorded from both limestone and iron caves, in both cases the caves are separated by large distances and the lithologies are disjunct. This incongruent and disjunct distribution is an indication of potentially unrecognized cryptic or overlooked species. This is more relevant when considered that ferruginous rock represents only 0.15% of the Brazilian territory (carbonatic rock 3.1%), nearly 10,000 km 2 (carbonatic rock 260.800 km 2 , Brazilian territory 8.516.000km 2 ) 23 , and www.nature.com/scientificreports/ that most of the biospeleological research is focused on large caves, usually in limestone 6,18,19 . The high richness of species restricted to small shallow caves, indicates that MSS plays a role as an extension of the cave environment. The State of Minas Gerais is the most diverse with 40 species of cave Collembola, the complex mosaicist lithology and the ecotone Cerrado Forest-Atlantic Forest are the main barriers associated to the richness of species restricted to caves and MSS. In this State, the iron rock subterranean habitats host 29 troglobites and provide habitat and refuge to 11 known troglophiles species.
For caves in non-ferruginous lithologies, the size and number of entrances influence the species richness by giving the surface fauna access to the subterranean environment, and as a sink for organic matter input 5 . Contrastingly the caves in iron rock are small and shallow, often with few meters of horizontal development, the connections to the MSS are conspicuous and abundant, providing a rather continuous subterranean habitat. In this context, instead, the distribution of the troglobitic species suggests that the entrances of iron rock caves are the limits of the available subterranean habitat for troglobites inside-out, and of suitable habitats for troglophiles outside-in (Fig. 1). We can consider the entrances of these caves as windows of the MSS, the spatial limit of the subterranean environment which presents the minimum conditions to the survival of a troglobite, while partially inhibits the dispersion of troglophiles deeper in the MSS. Troglobites can disperse underground more efficiently than troglophiles, however troglophiles are more efficient than troglobites to disperse through the surface 22 . In ferruginous lithology the size of the cave and its entrance influences the species richness 5 , mainly because large iron caves can greatly affect the capacity of collectors and biologists to access the troglobitic fauna in the MSS, as the number of accessible connections to the MSS increases exponentially with the length of the cave in iron rock 12 .
Another contrast of ferruginous rock caves is that the biotrophic flow seems to be inverted ( Fig. 1), in limestone caves the energy and fauna come from outside mostly through the cave entrance, and the fauna eventually speciate to become troglobitic, possibly restricted to the depths of a single cave. Despite the demonstrated existence of an epikarst, the particularities of the weathering process, the water percolation 24 , and different subterranean habitats as scree slopes and MSS 7,11 , limestone caves tend to be large and grow deep through dissolution of the rock by water during the genesis of the cave. The deeper the cave is, the lesser the permeability of the rock, the epikarst usually reaches about 15 m deep 24 .
In iron caves the fauna comes from the above ground through the MSS connections between surface and subterranean environments, the same happens with energy that comes with roots that reach the MSS abundantly 12 . The troglobites develop in the MSS and eventually reach the caves where it can be seen in its distribution limits, and the troglophiles go in the opposite direction, inhabiting the surface and going inside the caves to refuge from climate, but not going too far in the MSS (see Table 1, species marked with 1,3 ). Ferruginous rock-small and shallow caves, abundant roots, reticulated MSS; fauna and energy come mostly from the above ground (solid red arrows), troglobites inhabit the MSS and reach the deep limits of the cave horizontally, and lower limits of the soil vertically; troglophiles inhabit the surrounding and the cave, eventually reaching shortly in the MSS horizontally, but overlapping the troglobitic limits in the MSS and lower limits of soil vertically (dotted red arrows). (B) Limestone rock-large caves, usually not reached by roots, sparse or absent MSS; fauna and energy come largely through the cave entrances (solid red arrows), troglobites inhabit the deep aphotic zone reaching the aphotic intermediary zone horizontally, not reaching the upper MSS and epikarst vertically; troglophiles inhabit the surroundings and the cave, eventually reaching the deep aphotic zone horizontally, sometimes restricted to the MSS and epikarst vertically (dotted red arrows). Yellow to black bar represents the light reach. www.nature.com/scientificreports/ Pseudocryptic diversity. Large caves with deep aphotic zones, stable abiotic conditions, water pools, often hosting bat colonies, are correlated to high number of restricted species 12 , usually displaying classic troglomorphism as absence of eyes and body pigments, elongated appendages, increased body size 25 . In the ferruginous rock MSS the same troglomorphisms are present in most species, even though, we observed that some Entomobryoid Collembola are often reduced in size, with normal or shortened (even though always functional) appendages, similar to that of euedaphic fauna. Cryptic species recognized from a single widespread species complex through barcode sequencing, revealed related morphological differences corresponding to the species separation 26 . To access this information it is necessary to expand the morphological refinement, some cryptic species are grouped together as result of limited selection of diagnostic characters. This is by definition pseudocryptic species, when "individuals can be identified from morphology providing sufficient care is taken, but are so similar that there is a high probability of misidentification, even by a competent scientist" 27 .
Whether we accept that cryptic diversity in Collembola cannot be explained by accelerated rates of molecular evolution 28 , it is likely that the diversity of subterranean Collembola in ferruginous MSS and caves, results of the combination of the effects of lithology arrangement, phytophysiognomy and climate fluctuation at local scale.
Finally, the recognition of cryptic or pseudocryptic species within presumed widespread allopatric species is crucial to efficiently develop management and conservation plans 22  , which presented an overall species richness of 28 and 22 troglobites, respectively. These two caves are under different impact pressures, the former is in a protected area with controlled access, and the latter is under intense touristic exploitation 3 .
Myers et al. 1 combined richness, endemism, distribution spam and threats to the area to define places of priority for conservation, called hotspots. The number of troglobites, with a full consideration of the threats or conservation conditions of the caves and surroundings was, also, recently used as criteria for defining hotspot 3,6 .
The ferruginous rock outcrops in Brazil are under a intense economic pressure, the mining industry represents an important part of the production of commodities as iron ore and steel. The high diversity and endemism of cave Collembola found in recent studies (Table 1), affecting directly the beta diversity of the areas considering the species are found nowhere else, and the continuous threat to the subterranean habitats formed in ferruginous rock, justify categorizing the ferruginous subterranean habitats as hotspot for cave Collembola in the State of Minas Gerais. It is important to remark that the diversity considered here is only for Collembola species, and that the studies mentioned above have a much higher phylogenetic diversity.
Conservation policy implications. The ferruginous caves and the MSS represent sites of intense overlooked pseudocryptic diversification. Katz et al. 22 observed that for Collembola in limestone areas the detection of short-range endemics, genetic isolation, and apparent cryptic diversity has major conservation implications.
The results we present here bring several considerations on conservation strategies and policies. The high diversity and endemism rate observed for cave Collembola, associated to threats to the subterranean environments as mining, deforestation, and urbanization flag these areas as maximum priority and interest for planning putative conservation areas 29 . These areas demand a multi-factor approach to successfully develop policies which optimize the diversity conservation, particularly subterranean diversity.
Brazilian legislation has protective measures for caves, but allows the complete suppression of a cave for mining or other exploratory purpose, under a process for licensing the proposed activities. Even though some criteria are imposed, it fails in considering some important aspects of the cave structure in different lithology 12 . Under this perspective the whole extension of ferruginous (and carbonatic) rock deposits in Brazil are available for exploitation, with irreversible impact on the subterranean fauna. There is over than 9400 companies in activity in the country, producing about 235.000.000 ton/year of iron ore, the second biggest production in the world. More than 72% of the Brazilian iron ore reservoirs is located in the state of Minas Gerais, the locality of occurrence of 37 out of the 44 known species of Collembola found in ferruginous subterranean habitats in Brazil (Fig. 2).
Here we observed that the whole process needs a revision when comes to ferruginous rock, where the cave may not be the important spatial unit to preserve, instead, the high subterranean diversity areas must be surveyed, not only in caves but also in the MSS. It is possible that in some cases to protect a hill that harbors a thick layer of Canga with a troglobitic species rich MSS, would result more effective to preserve restricted subterranean fauna, than to protect a small and shallow cave with reduced troglobitic richness.
The state of Minas Gerais has 75 integral conservation units (defined by law), with maximum protection policy, however, these conservation units represent only 1.05% (~ 619,800 ha) of the state territory. There are other categories of conservation units, called of "sustainable use", with much less restrictive policies. These categories of conservation units are much less effective to preserve epigean species, due to the diverse usages and practices  30 . This procedure, implemented in the process for licensing new high impact exploratory activities, can improve the conservation effectiveness of the conservation units and compensation areas, precisely define the role of the cave in the conservation plan, and shift the focus towards troglobitic species richness.

Conclusions
Our results depict the ferruginous subterranean environment as an important hotspot for cave Collembola in the state of Minas Gerais, corroborating the expected high species richness in ferruginous rock caves and MSS. We also demonstrate that access pseudocryptic diversity as observed in the genera Arrhopalites, Pararrhopalites, Pseudosinella and Trogolahysa is mandatory for planning the conservation strategies for subterranean Collembola. The distribution of the species through the MSS can be favored by sustainable use conservation units, whether this fauna is surveyed along the licensing process. Finally, we conclude that the conservation planning for future conservation unit establishment must focus not only on caves but also in the MSS, accessing the fauna through sampling in prospection drilling holes. Protecting an area with high richness of endemic troglobites down in the MSS may be more effective than to protect a shallow cave when it comes to preserve troglobitic diversity.

Methods
Pseudocryptic diversity. The richness was the measure of the subterranean diversity, we surveyed all data about previous records for Brazilian Collembola cave species, ecological status, lithology, and distribution from the literature, and included 11 newly found pseudocryptic species from subterranean habitats in iron and limestone rock. The pseudocryptic species were verified by comparison of chaetotaxy and "micro-morphology" through optic and scanning microscopy of disjunct populations of a widespread morphotype. The imagery was compared under hypotheses of chaetotaxic and morphologic homology, previously defined by different authors. Those populations with consistent discrete chaetotaxic and morphologic patterns were assumed to be independent species, therefore they were taxonomically diagnosed, named, and ordered in a dichotomic identification key with all Brazilian species of the genus.
Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 14D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 24 external and 25 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.29 (holotype).
Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 20D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 70 external and 30 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.33 (n = 5).
Legs. Trochanteral organ triangular shape with about 19-21 spine-like chaetae, plus two psp one external and one on distal vertex of Omt (Fig. 26A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with two teeth , basal pair unequal, b.p. larger than b.a.; m.t. and a.t. absent.  Unguiculus with all lamellae smooth and lanceolate (a.i., a.e., p.i., p.e.) (Fig. 26B); ratio unguis: unguiculus = 1:   (Fig. 26C). Anterior side with seven ciliate, apically acuminate chaetae, three proximal, two subdistal and two distal mac; lateral flap with nine chaetae, four ciliate in the proximal row and five smooth in the distal row.
Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with five ciliate chaetae (two inner mac) and three psp (Fig. 26D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 35 external and 26 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.39 (n = 5).
Etymology. Species named after Dr. Felipe N. Soto-Adames for his contribution on Collembola taxonomy and systematics.
Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 29D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 40 external and 22 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.23 (holotype).
Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with five ciliate chaetae (three inner mac) and three psp (Fig. 32D). Dens posterior face with two or more longitudinal rows of spineslike chaetae about 22 external and 37-39 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.33 (holotype).
Etymology. Refers to the Baroque art (which is "barroco" noun, in Portuguese) of Mariana, Minas Gerais, type locality.
Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with five ciliate chaetae (two inner mac) and three psp (Fig. 35D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 60 external and 34 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.30 (holotype).
Etymology. Epitychia from Greek means success, in allusion to the collection site where the species was found São Sebastião do Bom Sucesso.
Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 38D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 30 external and 23 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.29 (n = 5).
Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with five ciliate chaetae (three inner mac) and three psp (Fig. 41D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 18 external and 24 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.26 (holotype).

Data availability
The datasets generated or analyzed during the current study are available from the corresponding author upon reasonable request.